Beta-galactosidase (EC3.2.1.23) is an enzyme that hydrolyzes the β-D-galactoside bond to release D-galactose, and, in general, it is widely distributed in microorganisms, and animals and plants. Beta-galactosidase is also referred to as lactase, and has been used as an enzyme for the production of a whey syrup from whey that is by-produced during the production of low-lactose milk for lactose intolerance or cheese, or as an active ingredient for medicines or supplements in patients with lactose intolerance. In addition, β-galactosidase has an ability to transfer the galactoside bond, and a method to prepare galacto-oligosaccharides (oligosaccharides with galactose residues) using this ability is known. Beta-galactosidases from a koji bacterium (Aspergillus oryzae), a yeast (Kluyveromyces lactis, Kluyveromyces marxinus), and a bacterium (Bacillus circulans) are known for use in these applications.
Among these, β-galactosidase derived from Bacillus circulans has been studied by Mozaffer et al. (non-patent document 1), Vetere et al. (non-patent document 2), and Ito et al. (non-patent documents 3 and 4). According to the non-patent document 1, purification of two kinds of enzymes each having a molecular weight of 240 kDa and 160 kDa is reported. It is further reported that the former has a high hydrolyzing activity, and the latter has a high transgalactosylation activity, and that the former showed a higher hydrolyzing activity against a synthetic substrate p-nitrophenyl-β-D-galactopyranoside (ONPG) than against lactose. On the other hand, according to the non-patent document 2, purification of three kinds of enzymes each having a molecular weight of 212 kDa, 145 kDa, and 86 kDa is reported. However, mutual protein chemical correlation and molecular biological characteristics (genetically) of these plural enzymes were not clear. In addition, in the non-patent documents 3 and 4, a gene cloning of the 67 kDa enzyme, and properties of the recombinant protein are reported, but the enzyme is specific to the β-1,3 bond, and does not act on the β-1,4 bond that is a bond of lactose present in a milk. Therefore, such an enzyme is different from the β-galactosidase ordinarily used in the treatment of milk or lactose derived from milk. In addition, two kinds of β-galactosidase genes derived from Bacillus circulans are registered at the GENBANK™ (GENBANK™ accession number of L03424 and L03425), but only gene sequences for these have been reported and it is not certain whether such genes encode actually an active protein.